Metallothioneins (MTs) are a class of ubiquitously occurring low molecular weight cysteine- and metal-rich proteins containing sulfur-based metal clusters. The conservation of these clusters in an increasing number of three-dimensional structures of invertebrate, vertebrate and bacterial MTs signifies the importance of this structural motif. It is becoming increasingly clear that mammalian MTs have diverse functions including involvement in zinc homeostasis, protection against heavy metal toxicity and oxidative damage (Vasak et al., 2005). Mammalian MTs are single chain polypeptides of 61, 60 or 68 amino acid residues with an N-terminal acetylmethionine and often alanine at the carboxyl terminus. They contain 20 cysteine residues, which are central to the binding of metals. MTs have characteristic C-X-C, C-Y-C, and C-C sequences, where X and Y are non-cysteine amino acids. There are 7 bivalent ions for every 20 cysteines forming metal thiolate complexes (7-10 g atoms of metal per mol MT in a two domain structure (Hussain et al., 1996).
There are four MT subgroups, namely MT1, MT2, MT3, and MT4. The MT1 and MT2 isoforms, which differ by only a single negative charge, are the most widely expressed isoforms in different tissues. Human MT genes are clustered at a single locus on chromosome 16, and at least 14 of the 17 genes so far identified, are functional. These encode multiple isoforms of MT1 (MT1A, B, E, F, G, H, I, K, L and X), MT2, MT3 and MT4 (Miles et al., 2000).
Stimuli that can induce MT expression are metals. hormons (e.g. glucocorticoids), cytokines, a variety of other chemicals, inflammation, and stress. MT degradation takes place mainly in the lysosomes. MT appears less susceptible to proteolysis in the metal bound state. In vivo, metal-MTs have far longer half-lives than apo-MT (Miles et al., 2000).
MT1 and MT2 are present throughout the brain and spinal cord, and that the main cell type expressing these MT isoforms is the astrocyte; nevertheless, MT1 and MT2 expression was also found in ependymal cells, epithelial cells of choroid plexus, meningeal cells of the pia mater, and endothelial cells of blood vessels (Hidalgo et al., 2001).
MTs are stress-inducible proteins that maintain metal homeostasis and scavenge free radicals. It is generally accepted that the major functions of MTs are related to metal metabolism. Postulated functions include detoxification and storage of heavy metals and the regulation of cellular copper and zinc metabolism in response to dietary and physiological changes. Because astrocytes, as well as ependymal cells, richly express MTs, an attractive hypothesis is that both cell types serve to protect the CNS from metals transported parenchymally from the blood or the cerebrospinal fluid. In AD subjects cerebral white matter contains numerous MT1- and MT2-expressing astrocytes with an intense immunoreactivity of the cell body (Zambenedetti et al., 1998). Chronic inflammation has been postulated raising the possibility that the etiology of AD has an immunological component. Cytokines and interleukin (IL)-1, for instance, elevated in AD, induce MT1 and MT2 production in astrocytes suggesting that these proteins may have a relevant role in providing long-term protection against oxidative damage, injury and inflammation with a multiple compensatory mechanism involving the osmotic regulation of some metal ions. Clear-cut effects of CNS injury on MT1 and MT2 expression were investigated. These studies have shown a dramatic induction of these MT isoforms in response to kainic acid-induced seizures, cryogenic injury, ischaemia, and after treatment with 6-aminonicotinamide (Penkowa et al., 1995; 2000; 2001). MT1 and MT2 are significant inhibitors of apoptotic cell death in the CNS (Giralt et al., 2002). MT1 and MT2 deficient mice showed both increased oxidative stress and neuronal apoptosis during epileptic seizures, experimental autoimmune encephalomyelitis (EAE), and following traumatic brain injury. Likewise, transgenic MT1 overexpressing mice showed significantly reduced oxidative tissue damage and cell death during traumatic brain injury, focal cerebral ischemia, and 6-aminonicotinamide (6-AN)-induced brain stem toxicity. Furthermore, MT1 and MT2I improve the clinical outcome and reduce mortality in different CNS disorders (Penkowa, 2002). MT has recently been shown to mediate neuroprotection in genetically engineered mouse model of Parkinson's disease (Ebadi et al., 2005).
MT2 treatment has recently been shown to significantly stimulate neurite extension from both dopaminergic and hippocampal neurons. Moreover, MT2 treatment significantly increases survival of dopaminergic neurons exposed to 6-hydroxydopamine (6-OHDA) and protects significantly hippocampal neurons from amyloid β-peptide-induced neurotoxicity (Køhler et al., 2003). Treatment using MT2 and other MTs has been suggested for motor neuron disease, head injury, Alzheimer's and Parkinson's diseases (WO03105910). The molecular mechanisms of neuritogenic and neuroprotective actions of MTs are so far unknown.
Recently, it has been shown that MT1 binds to low-density lipoprotein receptor related protein 2 (LRP2)/megalin and the corresponding binding site in MT1 has been identified (Klassen et al., 2004).
Megalin/LRP2 is a scavenger receptor due to its multifunctional binding properties. Among its ligands are lipoproteins, vitamin-binding and carrier proteins, drugs, hormones and enzymes as well as signalling molecules. The intracellular domain of megalin interacts with signalling adaptor molecules which has been shown to be involved in regulation of edocytosis (see for review May et al, 2005). However, it remains to be uncertain, whether megalin participates directly in cellular signalling cascades by transducing extracellular signals to intracellular binding partners.
One of the best-characterized physiological functions of megalin is the proximal-tubular reuptake of low-molecular weight proteins (Zou et al., 2004). Another permanent feature is that megalin is required for a proper forebrain development: megalin knock-out mice demonstrate holoprosencephaly (Willnow et al., 1996).